Plasma gas-synthesized 25-nm Ag-NPs were a kind gift from Dr. Karl Martin (Novacentrix, Austin, TX). Colloidal Ag-NPs at 10 nm and Au-NPs at 10 or 30 nm were synthesized by Dr. Steven Oldenburg (NanoComposix, San Diego, CA). Polysaccharide-coated Ag-NPs (PS-Ag) 10 and 25 nm were a generous gift from Dr. Dan Goia (Clarkson University, Center for Advanced Materials Processing, Potsdam, NY). These PS-Ag NPs were synthesized by the reduction of Ag ions in solution by a polysaccharide (acacia gum), which leads to a polysaccharide surface coating 8. All NPs were well characterized in our laboratory and were diluted in deionized water to 1 mg/ml, sonicated with a probe sonicator, and then further diluted in PBS or cell culture media.

Vero cells were obtained from the ATCC (CCL-81; Manassas, VA). The Vero cells were maintained in Dulbecco's modified Eagle's Medium (DMEM; Biowhitaker, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (PS; Invitrogen, Carlsbad, CA).

Vero cells were seeded into 96-well tissue culture-treated plates at a concentration of 50,000 cells/well. Twenty-four hours post-seeding, the Vero cells were exposed to Ag- or Au-NPs. The NPs were diluted to the various doses in DMEM + PS and sonicated for 30 s prior to exposure. At 24-h post-exposure, a standard MTS assay (Promega, Madison, Wisconsin) measuring mitochondrial function was performed to determine cell viability.

Vero cells were treated with the above NPs at various doses and were incubated for 24 h at 37°C in 5% CO₂. The NPs were removed and excess NPs were washed off with PBS. CV-(RR)₂ or CV-(FR)₂ reagents were added to the treated cells (diluted 1:250 in PBS) to detect changes in cathepsin B or cathepsin L activity, respectively (Biomol International, Plymouth Meeting, PA). Biomol's cathepsin detection kit utilizes the fluorophore, cresyl violet (CV), linked to two peptides moieties (arginine-arginine for cathepsin B and phenylalanine-arginine for cathepsin L), which are cleaved when the enzyme is active to release the fluorophore. The cathepsin substrate was incubated with the cells for 45 min at 37°C in 5% CO₂ and was then read using a fluorometer (BioTeck Synergy HT with Gen5 software) at excitation/emission wavelengths of 594/625 nm. Alternatively for the imaging, Hoechst (Molecular Probes – Invitrogen, Carlsbad, CA) was added directly to the CV-(RR)₂ or CV-(FR)₂ reagents (1:1,000), and the cells were imaged following the 45-min incubation with the Becton Dickinson Pathway 435 spinning disc Confocal microscope (BD, Franklin Lakes, NJ). The CV-(RR)₂ or CV-(FR)₂ reagents were detected with a rhodamine filter with settings normalized to the control (untreated) sample.

Purified cathepsins B and L were purchased from Biomol International (SE198 and SE201, respectively). Three micrograms of each protein was incubated with the 4 types of Ag-NPs at either 10 or 50 μg/mL in 300 μL sterile H₂O for 1 h at room temperature with rotation. Following this incubation, the mixture was added in triplicate to a 96-well black-sided plate (100 μL/well). Hundred microliters of the respective Biomol CV reagent (diluted 1:125 in water) was then added to each well of the enzyme-NP mixture. The reaction was incubated for 45 min at 37°C in 5% CO₂ and was then read in a fluorometer. The process was repeated to obtain a total of six replicates.

Data were expressed as the mean ± standard error of the mean (SEM). The one-way ANOVA (Prism 5 statistical and graphing software; GraphPad Software, Inc, La Jolla, CA) was used for the data analysis. The one-way ANOVA was used to determine the effect of test compound concentration on the mean number of treatments/well. P ≤ 0.05 was used as the level for significance.

After a 24-h exposure, a 25% decline in cell viability was observed in Vero cells exposed to 50 μg/ml of 10-nm uncoated Ag-NPs (Figure 1). Treatments with 10-nm uncoated Ag-NPs at 75 and 100 μg/ml resulted in a 60% reduction in cell viability (Figure 1). There was no further reduction in cell viability in the 50 μg/ml dose, but the cells treated with 75–100 μg/ml died off by day 2 (data not shown). Concentrations of uncoated 10-nm Ag-NPs lower than 50 μg/ml had little effect on Vero cell viability (Figure 1). The 10-nm PS-Ag had no significant effects on the Vero cells in the first 24 h (Figure 1), but the 75 and 100 μg/ml doses demonstrated a 25% reduction in viability after 48 h (data not shown), suggesting an instability of the coating. The concentrations of Ag-PS 10 nm at 50 μg/ml or less had no effect on cell viability at later time points (data not shown). There was little cytotoxicity observed in Vero cells treated with the uncoated or polysaccharide-coated 25-nm Ag-NPs (Figure 1).

A significant decrease in red fluorescent intensity, indicating a decrease in cathepsin B activity, was observed in the 50 μg/ml doses of 10 nm both uncoated and PS-coated and 25-nm uncoated Ag-NPs (Figure 2d, f, h) over the untreated control (Figure 2b). There was little visual difference in red fluorescence intensity between the 10 μg/ml treated groups (Figure 2c, e, g, i) and the 25-nm PS-Ag at 50 μg/ml (Figure 2j) from the untreated control (Figure 2b), although the 10-nm PS-Ag and 25-nm uncoated Ag-NPs did have a significant decline in fluorescence intensity (Figure 2 table). The decrease in cathepsin B activity in Vero cells treated with Ag-NPs was confirmed via fluorescent quantification in a fluorescent plate reader and a dose-dependent decrease in cathepsin B activity is observed in all treatment groups except for the 25-nm PS-Ag, which interestingly had no effect on cathepsin B activity (Figure 2 table).

Cathepsin L activity appears to be more sensitive to Ag-NP exposure. All 4 types of Ag-NPs tested demonstrated a significant reduction of cathepsin L activity in Vero cells (Figure 3). Minimal cathepsin L activity was observed when the cells were treated with any of the Ag-NPs at 50 μg/ml (Figure 3d, f, h, j), and this decrease was determined by quantitative assessment to be statistically significant (Figure 3 table). There was little discernable difference in red fluorescence between the 10 μg/ml uncoated Ag-NP-treated cells (Figure 3c, g) versus the untreated control (Figure 3b), however, the PS-Ag-NPs (10 and 25 nm) appear to cause a slight, yet significant decline in cathepsin L activity at this dose (Figure 3e, i, table). The decrease in cathepsin L enzymatic activity caused by Ag-NPs was much greater than that observed with cathepsin B (Figures 2, 3).

To determine whether the Ag-NPs were influencing the cell signaling or the enzyme directly, the Ag-NPs were also incubated with purified cathepsin B or L, which were then assayed for activity. A significant decrease was seen in the activity of the purified cathepsin B enzyme with the 10-nm Ag-NPs at 10 μg/ml and even greater at 50 μg/ml, regardless of the presence or absence of the PS coating (Figure 4a). The 25-nm uncoated Ag-NPs had little effect on cathepsin B activity at 10 μg/ml, but exhibited similar affects as the 10-nm particles at a dose of 50 μg/ml (Figure 4a). The 25-nm PS-Ag displayed a similar effect as the 25-nm uncoated Ag-NP, with no effect at 10 μg/ml and a significant decrease in cathepsin B activity at 50 μg/ml, however, the decrease with this particle was much less than that with the uncoated 25-nm Ag-NP (Figure 4a). When the purified cathepsin L enzyme was treated with Ag-NPs, the effect on activity was also much more significant than the effect on purified cathepsin B (Figure 4). All four Ag-NPs tested caused a significant decrease in cathepsin L activity at both the 10 and 50 μg/ml doses, with the 10-nm Ag-NPs (uncoated and PS-coated) causing a more pronounced effect (Figure 4b).

The Au-NPs at 10 and 30 nm had minimal effects on the viability of Vero cells in culture (Figure 5). There was a slight decrease in the mitochondrial function of the Vero cells treated with very low (5 μg/ml) or very high (100 μg/ml) doses of Au-NP 10 (Figure 5), but a recovery was made by the cells following a 48-h exposure (data not shown).

After a 24-h exposure of Vero cells to Au-NPs, a slight decrease in cathepsin B activity was observed in cells treated with either 10- or 30-nm Au-NPs at doses of 5–25 μg/ml (Figure 6a). A more significant decrease in cathepsin B activity was observed when the Vero cells were exposed to higher doses of these NPs (Figure 6a). Conversely, the low doses of 10- and 30-nm Au-NPs actually had a stimulatory effect on cathepsin L activity (Figure 6b). At concentrations of 1–10 μg/ml of the 10-nm particles and 5–50 μg/ml of the 30-nm particles, a significant upregulation of cathepsin L was observed in Vero cells (Figure 6b). A significant decrease in cathepsin L activity was finally observed at 50–100 μg/ml of the 10-nm Au-NP, but no decrease was ever seen in Vero cells treated with 30-nm Au-NPs (Figure 6b). Interestingly, incubation of the Au-NPs with the purified cathepsin enzymes had little impact on the enzymatic activity of either cathepsin B or L at 10 μg/ml (Figure 6c, d), and although there was a significant decrease in the 50 μg/ml dose of Au-NPs (Figure 6c, d), the decline in cathepsin activity with these NPs was not as dramatic as that observed with the Ag-NPs (Figure 4).